Determination of Nine Phenolic Components in Leaves ofCrataeguspinnatifida Bge.

Supported by Project of Natural Science Foundation of Hebei Province (C2015204187).

1 Introduction

Crataeguspinnatifida Bge.is a member of family Rosaceae. As a traditional Chinese medicinal material, both dried berries and leaves have been included in VariorumofShennong’sClassicofMateriaMedicaandHandbookofPrescriptionsforEmergencies[1]. Modern pharmaceutical researches on the leaves of Crataegus started from the systematic study undertaken by the Federal Republic of Germany in the 1980s, and a standardized Crataegus extract was developed for the treatment of NYHA class I-II heart diseases[2]. In the end of the last century, some Chinese scholars developed flavonoid drug using C. pinnatifida Bge. leaves, and the drug was included in the ChinesePharmacopoeia[3]. At present, many scholars use Crataegus extract to apply to NYHA class I-II heart diseases[4]. C. pinnatifida Bge. leaves not only contain flavonoids, but also contain procyanidins, phenolic acids and other phenolic components[5-7].

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In recent years, some scholars analyzed chlorogenic acid, vitexin-4"-O-glucoside, vitexin 2"-O-rhamnoside, hyperoside, and isoquercitrin by HPLC[8-10], but not refer to procyanidins. Some European scholars also have developed methods for analysis of procyanidins in Crataegus spp. leaves of C. pinnatifida Bge.[11], but the methods could not analyze flavonoids. Using UPLC-MS/MS, Xu Wen etal.[12] determined flavonoids and proanthocyanidins in Tetrastigmahemsleyanum Diels et Gilg, but the method has a high requirement for the instrument and thus is not suitable for wide application. In this experiment, on the basis of previous researches of other scholars[13-14], we developed a method for determining nine phenolic components in C. pinnatifida Bge. leaves, to provide references for quality control of C. pinnatifida Bge. leaves and relevant products.

2 Materials and methods

2.1 Materials and instruments C. pinnatifida Bge. leaves included Dawang, Yubeihong, Ji’anzirou, Dahuo, Zhaikou, Xiaotangqiu, Fulihong, Datangqiu, Fengshouhong, Changkou, Qiujinxing, Dahuangmianzha, Liaohong, Damiaoqiu, Tianshui, Anzedaguo, Xifenghong, Laishuidajinxing, Jinhong, Zigenxi, Mopan, 9039, Mianhong, Xinglong 2, Xinglong 3, Xinglongzirou, Xiaojinxing, Wulinghong, and Yanranghong. All these leaves were collected from Xinglong County Hawthorn Resource Garden in Hebei Province on May 27, 2015. The samples were frozen and dried, stored in the vacuum dryer for use. Standard reference substances: epicatechin and chlorogenic acid bought from American Sigma Company; hyperoside, isoquercitrin, and vitexin 2"-O-rhamnoside were bought from Shanghai Yuanye Bio-Technology Co., Ltd.; procyanidin B2, procyanidin C1, procyanidin D1, and eucomic acid were isolated and extracted from the fruit of C. pinnatifida Bge[14]. Electrospray mass spectrometry, nuclear magnetic resonance (NMR), and thionolysis-HPLC were applied to identify the above compounds, and confirmed by comparison with related references[14]. The purity obtained by the peak area normalization method was higher than 95%. The purified water was product of Hangzhou Wahaha Group Co., Ltd. Acetonitrile and methanol were products of Honeywell Burdick & Jackson Company, chromatography reagent; other reagents were analytical reagents (AR).

Agilent-1200 high performance liquid chromatography system consists of the online degaser, quaternary low pressure gradient pump, photo-diode array detector, and Agilent Chemstation (Agilent Company); CO-3010 column temperature control cabinet (Tianjin Ameritech Scientific Corporation); KQ-5200E ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).

3.1 Comparison of chromatographic columns For ODS and BDS columns with the same specifications, the chromatographic peaks and resolution of the BDS column were better than that of the ODS column; the longer the number of chromatographic plates, the better the separation of procyanidin B2, procyanidin C1 and procyanidin D1 from other impurities.As a result Hypersil BDS C18 (250 mm × 4.6 mm id, 5 μm) was used as the experimental column.

2.2.1 Preparation of samples. Weighed 0.5 g of C. pinnatifida Bge. leaves and put into a mortar, dripped three drops of 80% phosphoric acid and added 15 mL pure water and ground evenly, transferred with 95% ethanol to 50 mL volumetric flask, extracted 11 min with ultrasonic wave, fixed the volume with 95% ethanol, metered volium, took the supernatant and centrifuged at 4 000 r/min for 5 min, filtered with 0.45 μm microporous membrane, for HPLC analysis.

2.2.2 Compare of HPLC analysis conditions. Selected three kinds of chromatographic columns Hypersil BDS C18 (250 mm × 4.6 mm id, 5 μm), Hypersil BDS C18 (200 mm × 4.6 mm id, 5 μm), and SinoChrom ODS C18 (200 mm × 4.6 mm id, 5 μm), three kinds of mobile phase (methanol-0.05% formic acid, acetonitrile-0.05% formic acid, methanol-acetonitrile -0.05% formic acid), and different gradient elution procedures to carry out the experiment.

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3.4 Determination of the content of nine phenolic components in leaves of different varieties ofC. pinnatifida Bge.

3 Results and discussion

2.2 Experimental method

3.2 Selection of mobile phase If using methanol-0.05% formic acid as the mobile phase, when 40% methanol solution was used, ten kinds of polyphenols could not be separated well; when the concentration of methanol reduced to 25%, the retention time of the procyanidin B5 was longer. If using acetonitrile -0.05% formic acid as the mobile phase, when the acetonitrile solution was 20%, the peak time of procyanidin B5 was earlier, but the other components were not well separated; when the acetonitrile content redued to 12%, the other components were well separated. When using methanol-acetonitrile-0.05% formic acid as the mobile phase, in order to save time, gradient elution was carried out with methanol and acetonitrile 1∶2 (V/V) as solution A and water as solution B. When the samples were analyzed using the HPLC conditions in Section 2.2.3, the separation of the target compounds was better and the baseline was stable. The chromatograms of nine phenolic components and samples of C. pinnatifida Bge. leaves are shown in Fig.1. According to Fig.1, the nine polyphenols in the samples were well separated from the other peaks within 47 min under the selected analysis conditions.

Note: 1. Eucomic acid; 2. Chlorogenic acid; 3. Procyanidin B2; 4. Epicatechin; 5. Procyanidin C1; 6. Procyanidin D1; 7. Vitexin 2"-O-rhamnoside; 8. Hyperoside; 9. Isoquercitrin.

Fig.1 High performance liquid chromatograph for reference substance of phenolic components (A) and sample ofCrataeguspinnatifida Bge. (B)

3.3.2 The regression equation, linear range and limit of detection of the method. The standard reference solution was prepared in accordance with the method in Section 2.2.4, statistics were made for the peak area of each standard reference substance. Through calculation, we obtained the regression equation, correlation coefficient and linear range. The limit of detection was obtained with three times the signal-to-noise ratio. The results were listed in Table 2. The results showed that there was a good linear correlation between response value and injection volume, the correlation coefficient was in the range of 0.999 5-0.999 9, the limit of detection was in the range of 1.0-1.5 μg/mL, which can meet the requirement of quantitative analysis of routine samples.

Using the method in Section 2.2.3, we analyzed and detected the content of nine phenolic components in leaves of 29 varieties of C. pinnatifida Bge. and the results are listed in Table 4. for phenolic acids and flavonoids, as well as in Table 5 for procyanidins.

德国水兵俱乐部旧址的1层门厅以及走廊铺设红棕与米黄相间的几何方块釉面砖，尺寸为152 mm×152 mm(图4).此种地砖成本相对较低，因为每片地砖只涉及一种类型的黏土和颜色；同时几何形地砖易于装饰边框，创作各种形状的地面设计.湖南路23—25号波特尔公寓旧址在入口处与楼梯间地面也使用了同样规格的黑、黄色方块釉面砖.

Table 1 Evaluation of the method precision

ComponentRSD∥%Eucomicacid1．80ProcyanidinB21．31Epicatechin0．94ProcyanidinC11．01ProcyanidinD11．85Chlorogenicacid1．96Vitexin2＂⁃O⁃rhamnoside0．80Hyperoside1．00Isoquercitrin0．83

3.3 HPLC method evaluation

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2.2.3 HPLC analysis conditions. Chromatographic column: Hypersil BDS C18 (250 mm × 4.6 mm id, 5 μm); mobile phase A∶methanol/acetonitrile = 1∶2 with 500 μL/L formic acid; gradient elution procedure: 0-26 min, 8%-20% A; 26-30 min, 20%-50% A; 30-35 min, 50% A; 35-37 min, 50% A; 37-40 min, 8% A; flow rate: 0.8 mL/min; column temperature 45℃; the injection volume: 10 μL; DAD detector, detection wavelength: procyanidin B2, procyanidin C1, procyanidin D1, and eucomic acid adopted 280 nm, chlorogenic acid, hyperoside, isoquercitrin, and vitexin 2"-O-rhamnoside adopted 350 nm, and quantitative analysis was made using external standard peak area method.

Table 2 The regression equation, correlation coefficient, linear range and limit of detection of standard reference substance

ComponentRegressionequationCorrelationcoefficientLinearrange∥μg/mLLimitofdetection∥μg/mLEucomicacidY=1．1981X-3．61360．99952-5001．5ProcyanidinB2Y=3．6745X+20．30．99962-5001．0EpicatechinY=3．8049X+27．1900．99982-5001．0DChlorogenicacidY=8．150X+29．6100．99972-5001．0Vitexin2＂⁃O⁃rhamnosideY=9．641X+20．7890．99992-5001．0ProcyanidinC1Y=6．1326X-14．5700．99982-5001．5ProcyanidinD1Y=4．9856X-2．56920．99992-5001．5IsoquercitrinY=3．0048X-9．880420．99962-5001．5HyperosideY=1．5974X-5．24780．99972-5001．5

3.3.3 Recovery rate of spiked samples. In C. pinnatifida Bge. leaf samples, added the accurately weighed nine standard phenolic samples, treated them in accordance with the method in Section 2.2.1, divided them into five parts in parallel, measured the non-spiked samples and spiked samples in accordance with chromatographic conditions in Section 2.2.3, made statistics of the peak area, calculated the recovery rate of spiked samples, and the results are listed in Table 3. According to the results, the recovery rate of spiked samples of nine phenolic components was in the range of 93.7%-110.2%, RSD was in the range of 0.69%-4.58%, the recovery rate was high, which can meet the requirements for conventional quantitative analysis.

2.2.4 Plotting of standard curve. The chlorogenic acid, eucomic acid, epicatechin, procyanidin B2, procyanidin C1, procyanidin D1, vitexin 2"-O-rhamnoside, hyperoside, and isoquercitrin reference substances were dissolved in methanol, prepared into 0.5 mg/mL mixed reference stock solution, diluted with methanol to different concentrations of standard solution, analyzed and plotted the standard curve.

3.3.1 Precision. Accurately sucked up 0.5 mg/mL mixed reference substance, injected five times in accordance with chromatographic conditions in Section 2.2.3, made statistics of the peak area of each chromatographic peak, the results are listed in Table 1. The relative standard deviation (RSD) was in the range of 0.80%-1.96%, showing that this method has a high precision.

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Table 3 Recovery rate of spiked samples of nine phenolic components

ComponentInjectionvolumemg/gRecoveryrate%RSD%Eucomicacid5．6096．21．53Chlorogenicacid2．71108．10．89ProcyanidinB21．30100．60．69Epicatechin2．8793．71．87Vitexin2＂⁃O⁃rhamnoside9．78110．21．17ProcyanidinC10．6595．60．71ProcyanidinD10．7298．71．51Hyperoside0．2696．43．52Isoquercitrin0．0799．04．58

Table 4 Content of phenolic acids and flavonoids in leaves of 29 varieties ofCrataeguspinnatifida Bge. (mg/g D.W)

VarietyEucomicacidChlorogenicacidVitexin2＂⁃O⁃rhamnosideHyperosideIsoquercitrinDawang13．2±0．042．60±0．1711．2±0．180．38±0．010．07±0．01Yubeihong6．29±0．350．95±0．0916．7±0．060．30±0．020．11±0．01Jianzirou13．3±0．233．06±0．0719．7±0．350．57±0．010．19±0．02Dahuo14．5±0．264．01±0．0914．2±0．330．25±0．010．05±0．01Zhaikou12．9±0．226．46±0．2017．0±0．450．74±0．010．17±0．01Xiaotangqiu8．05±0．221．83±0．1314．0±0．650．55±0．040．19±0．01Fulihong13．7±0．174．48±0．1315．6±0．220．36±0．010．03±0．01Datangqiu13．8±1．305．04±0．3516．1±1．570．51±0．050．11±0．01Fengshouhong13．4±0．101．83±0．104．64±0．070．16±0．010．03±0．01

(To be continued)

(Continued)

VarietyEucomicacidChlorogenicacidVitexin2＂⁃O⁃rhamnosideHyperosideIsoquercitrinChangkou12．7±0．503．52±0．1311．1±0．450．67±0．040．21±0．01Qiujinxing12．8±0．651．80±0．1613．1±1．111．25±0．130．28±0．01Dahuangmianzha9．89±0．531．17±0．0614．0±0．860．36±0．030．09±0．01Liaohong13．4±0．504．13±0．2915．8±0．110．53±0．010．13±0．01Damianqiu3．97±0．380．45±0．076．57±0．84TraceaTraceaTianshui13．5±0．533．25±0．1413．1±0．160．30±0．010．10±0．01Anzedaguo6．88±0．690．93±0．0920．7±1．230．43±0．040．11±0．01Xifenghong3．74±0．280．51±0．0216．0±0．290．24±0．010．06±0．01Laishuidajinxing10．7±0．272．37±0．1716．0±0．410．16±0．010．02±0．01Jinhong11．6±0．703．62±0．1412．1±0．380．41±0．030．11±0．01Zigenxi15．0±0．605．76±0．0212．9±0．260．88±0．030．28±0．01Mopan14．2±1．023．71±0．3320．9±0．950．55±0．040．09±0．01903913．4±0．273．89±0．1414．8±0．190．41±0．020．08±0．01Mianhong13．9±1．323．63±0．3424．9±2．100．72±0．050．23±0．02Xinglong29．48±0．462．44±0．0616．4±0．120．44±0．010．06±0．01Xinglong313．6±0．373．96±0．1719．3±0．230．36±0．030．06±0．01Xinglongzirou13．4±0．394．03±0．1617．7±0．300．42±0．010．11±0．01Xiaojinxing8．26±0．302．45±0．1115．1±0．080．48±0．020．06±0．01Wulinghong12．3±0．132．53±0．086．05±0．162．55±0．070．54±0．04Yanranghong17．3±0．644．16±0．3017．6±0．330．41±0．010．11±0．01Meanvalue11．6±3．233．05±1．4914．9±4．330．53±0．450．13±0．11

Table 5 Content of procyanidins in leaves of 29 varieties ofCrataeguspinnatifida Bge. (mg /g D.W)

VarietyEpicatechinProcyanidinB2ProcyanidinC1ProcyanidinD1Dawang2．48±0．192．24±0．042．39±0．101．98±0．19Yubeihong0．14±0．010．78±0．040．28±0．010．77±0．03Jianzirou1．90±0．132．14±0．101．44±0．061．18±0．11Dahuo1．63±0．111．95±0．051．00±0．071．03±0．03Zhaikou3．70±0．052．26±0．122．22±0．101．93±0．11Xiaotangqiu0．69±0．031．20±0．061．20±0．031．66±0．15Fulihong1．95±0．071．71±0．031．31±0．131．92±0．12Datangqiu2．81±0．282．13±0．191．71±0．151．62±0．16Fengshouhong2．51±0．221．38±0．051．46±0．130．99±0．12Changkou2．96±0．172．07±0．051．99±0．021．62±0．13Qiujinxing1．46±0．131．39±0．150．95±0．101．32±0．11Dahuangmianzha0．59±0．031．25±0．030．93±0．061．19±0．10Liaohong2．20±0．321．60±0．121．33±0．161．48±0．14Damianqiu0．12±0．020．78±0．020．19±0．020．76±0．08Tianshui1．70±0．131．78±0．121．24±0．131．39±0．11Anzedaguo0．18±0．030．92±0．100．41±0．040．73±0．04Xifenghong0．02±0．010．60±0．020．20±0．010．91±0．09Laishuidajinxing0．80±0．051．30±0．060．94±0．041．49±0．11Jinhong1．61±0．081．54±0．131．20±0．111．50±0．03Zigenxi3．64±0．032．32±0．122．31±0．051．67±0．10Mopan1．70±0．131．74±0．121．31±0．111．98±0．1890392．13±0．031．90±0．041．47±0．091．48±0．10Mianhong2．59±0．192．50±0．232．10±0．132．24±0．10Xinglong20．72±0．051．13±0．060．83±0．071．30±0．12Xinglong31．76±0．131．80±0．151．39±0．041．35±0．06Xinglongzirou1．20±0．071．70±0．041．39±0．041．21±0．10Xiaojinxing0．68±0．051．25±0．011．23±0．111．21±0．10Wulinghong1．19±0．102．02±0．052．23±0．062．27±0．05Yanranghong1．46±0．171．72±0．071．10±0．071．58±0．12Meanvalue1．61±1．001．63±0．501．29±0．601．44±0．41

According to Table 4 and Table 5, the eucomic acid, chlorogenic acid, and vitexin 2"-O-rhamnoside, hyperoside, isoquercitrin epicatechin, procyanidin B2, procyanidin C1 and procyanidin D1 were detected in leaves of 29 varieties of C. pinnatifida Bge. The content of hyperoside and isoquercitrin was relatively low, the mean value was 0.53 mg/g D.W and 0.13 mg/g D.W, and the content of hyperoside and isoquercitrin in Damianqiu was lower than the limit of detection. According to the detection results, we obtained the ratio of C-glycoside flavonoids (vitexin 2"-O-rhamnoside) to O-glycosides (C/O) was 22.6, and the content of eucomic acid was higher than chlorogenic acid in leaves of C. pinnatifida Bge., which was consistent with the results of the reference[5]. Epicatechin content in leaves of C. pinnatifida Bge. was high and the mean value was 1.61 mg/g D.W, which was consistent with the results of the reference[15]. The average content of the nine phenolic components was as follows: isoquercitrin, hyperoside, procyanidin C1, procyanidin D1, epicatechin, procyanidin B2, chlorogenic acid, eucomic acid, and vitexin 2"-O-rhamnoside. The flavonoids in leaves of C. pinnatifida Bge. include hyperoside, isoquercitrin, and vitexin 2"-O-rhamnoside, and the total average content was up to 15 mg/g D.W; the total average content of phenolic acids (chlorogenic acid and eucomic acid) was about 15 mg/g D.W; the total average content of procyanidins (procyanidin B2, procyanidin C1, and procyanidin D1) was about 6 mg/g D.W.

4 Conclusions

In this experiment, we established a reversed-phase high-performance liquid chromatography (RP-HPLC) for determination of nine phenolic components in leaves of C. pinnatifida Bge. In optimized chromatographic conditions, the nine phenolic components were well separated within 47 min, and the method is sensitive, accurate and reproducible, and can be applied for the quantitative analysis of phenolic components in leaves of C. pinnatifida Bge. Using the established method, we analyzed the content of nine phenolic components in leaves of 29 varieties of C. pinnatifida Bge. The results indicate that flavonoids and phenolic acids are the main components of polyphenols in leaves of C. pinnatifida Bge. The content of nine phenolic components is different in different varieties.

皇城相府分内城、外城，由一组别具特色的明清城堡式官宅建筑群组成，建筑依山就势，随形生变，鳞次栉比，雕梁画栋，斗拱飞檐，金碧辉煌。皇城相府集古代官宅民居、宗祠庙宇、园林书院、纪念性建筑和防御工事于一体，亦为四百年前农、商、学、官文化的一个缩影，堪称中国古代建筑文化的艺术瑰宝。

References

[1] SONG LR, HONG X, DING XL, etal. Modern Chinese medicine dictionary[M].Beijing: People’s Medical Publishing House，2001: 165-168. (in Chinese).

[2] WAGNER H. Phytomedicine research in Germany[J].Environmental Health Perspectives，1999，107(10) : 779-781.

[3] Chinese Pharmacopoeia Commission.Pharmacopoeia of the People’s Republic of China[S].Beijing: Chemical Industry Press，2005: 575-576. (in Chinese).

[4] LI T, ZHANG L, QI G. A clinical study on the treatment of NYHA Ⅱperiod of congestive heart failure using 350 hawthorn fruit standard extracts[J].Drugs & Clinic，2004，19(6) : 264-265. (in Chinese).

[5] GAO Z，JIA YN，CUI TY，etal.Quantification of ten polyphenols in the leaves of Chinese hawthorn(Crataeguspinnatifida Bge.var. major N.E.Br.) by high performance liquid chromatography[J]. Asian Journal of Chemistry，2013，25(18) : 10344-10348.

[6] LI BL, ZHAO L, SHEN YH, etal.Optimization of ultrasonic extraction of oligomeric proantho cyanidins from hawthorn leaves[J].Guangzhou Chemical Industry and Technology，2016，11(10) : 138-140. (in Chinese).

[7] XU HL. HPLC-DAD-ESI-MS /MS identification of the chemical constituents in hawthorn leaves and study on the preparation of three high purity flavonoid glycosides[D].Shanghai: The Second Military Medical University，2008. (in Chinese).

[8] DU YL, PAN HF. Determination of chlorogenic acid and vitexin rhamnoside in major hawthorn leaf of Chengde by HPLC[J].Journal of Shanxi Medical College for Continuing Education，2016, 26(1) : 4-7. (in Chinese).

[9] PAN WD, DU YL, ZHAO SN, etal. Simultaneous determination of 4 components in Chengde hawthorn leaf by HPLC[J]. Journal of Chengde Medical College，2015，32 (4) :279-281. (in Chinese).

[10] WANG LD, LI YR, ZHANG XF, etal. HPLC fingerprint study of hawthorn leaf[J].China Journal of Experimental Traditional Medical Formulae，2011，17(19) : 74-78. (in Chinese).

[11] GABRIELA ER，BEAT OS. Quantitative reversed-phase high-performance liquid chromatography of procyanidins in Crataegus leaves and flowers[J]. Journal of Chromatography A，1999，835(1-2) : 59-65.

[12] XU W, FU ZQ, LIN J, etal.Rapid simultaneous determination of ten major flavonoids in Tetrastigmahemsleyanum by UPLC-MS/MS[J]. Acta Pharmaceutica Sinica，2014，49 (12) : 1711-1717. (in Chinese).

[13] CUI T，LI JZ，KAYAKARA H，etal. Quantification of the polyphenols and triterpene acids in Chinese hawthorn fruit by high-performance liquid chromatography[J].Journal of Agriculturaland Food Chemistry，2006，54(13) : 4574-4581.

[14] KANG XH.Study onthe chromatographic retention behavious recovery rateof thiolysis of hawthorn procvanidins[D]. Baoding: Agricultural University of Hebei Province，2014. (in Chinese).

[15] LIU P，KALLIO H，YANG B. Phenolic compounds in hawthorn (Crataegusgrayana) fruits and leaves and changes during fruit ripening[J]. Journal of Agricultural & Food Chemistry，2011，59(20) :11141-11149.